A method for parallel microscale protein labeling and precise control over the average degree of labeling (aDoL)

A widely used approach for protein conjugation is through the lysine residues reacting with NHS- or other active esters. However, it is a challenge to precisely control the degree of labeling (DoL) due to the instability of active ester and variability of reaction efficiencies. Here, we provide a protocol for better control of aDoL using existing Copper-free Click Chemistry reagents. It is a two-step reaction with one purification in between. Briefly, proteins of interest were first activated with azide-NHS. After removing unreacted azide-NHS, the protein-N3 is then reacted with a limited amount of complementary click tag. Our studies have shown the click tag will fully react with the protein-N3 after 24 h’ incubation, and therefore does not require additional purification steps. As such, the aDoL is equal to the input molar ratio of the click tag and the protein. Furthermore, this approach offers a much simpler and more economical way to perform parallel microscale labeling. Once a protein is pre-activated with N3-NHS, any fluorophore or molecule with the complementary click tag can be attached to the protein by mixing the two ingredients. Quantities of the protein used in the click reaction can be at any desired amount. In one example, we labeled an antibody in parallel with 9 different fluorophores using a total of 0.5 mg of antibody. In another example, we labeled Ab with targeted aDoL value from 2 to 8. In a stability comparison study, we have found the conjugated fluorophore using the suggested click protocol stayed attached to the protein longer than with standard NHS-fluorophore labeling.

www.nature.com/scientificreports/ plus one-step purification. Nonetheless, due to the instability (hydrolysis) of the active ester, inconsistency of reaction efficiencies, and concerns of over-labeling affecting the protein's function 18 , it remains a daunting task.
Here, we describe a labeling method that guarantees targeted aDoL using readily available copper-free click chemistry reagents. The attachment sites are stochastically targeted to the lysine residues on the protein surface, and the aDoL is the average number of tag (fluorophores) conjugated to each protein. It is a two-step reaction with one purification step in between (shown in Fig. 1). As a first step, the protein was activated with N 3 -NHS and excess N 3 -NHS was removed after the reaction. In the second step, the protein-N 3 was mixed with fluorophore-DBCO at a molar ratio equivalent to the desired aDoL. After a 24-h incubation, the labeled protein is ready for use.
The success of the proposed labeling protocol relies on two conditions: (1) In the activation step, the average number of N 3 attached to the protein should be higher than the desired aDoL, (2) the added fluorophore-DBCO must be fully consumed in the second step, thus making additional purification unnecessary. The protocol was tested on two protein systems, a small protein: apomyoglobin (apoMb, 17 kDa) and an antibody (150 kDa). They were both labeled with AZ488-DBCO, achieving aDoL of 2-4 for apoMb and aDoL of 2-8 for antibody. Chromatography and Fluorescence Correlation Spectroscopy (FCS) were used to monitor the labeling reaction, detect unreacted fluorophores, and characterize the brightness of the conjugates.
FCS measures the signal fluctuation of fluorescent molecules as they freely diffuse through a well-defined illumination volume (~ 1 fL). This elegant technique was introduced more than 30 years ago 19 , but was not frequently used until the mid-90's when faster computers and advanced instruments became available. A comprehensive overview of the FCS basic principles and applications can be found elsewhere 20 . The calculated autocorrelation curve provides information on the diffusion rate of the fluorescent molecules. A smaller molecule (e.g., a free fluorophore) has a faster diffusion rate comparing to a fluorescently labeled protein and thus reflected in a rightshifted autocorrelation curve during the labeling reaction.

Methods.
AlexaFluor 488-SDP labeling. The anti-NGAL Ab1 at 2 mg/mL in PBS buffer was mixed with eightfold molar excess of AF488-SDP ester. The reaction was carried out at 2-8 °C overnight. Excess AF488-SDP were removed by passing the sample twice through Zeba spin desalting columns.
Pre-activation for click labeling. 1-2 mgs of N 3 -NHS was dissolved in DMSO to a final concentration of 10 mg/ mL. The concentration of N 3 -NHS was determined by accurately weighing or measuring the hydrolyzed product, NHS, ε 260 nm = 9700/M/cm 22 . Both methods were used to determine the concentration of N 3 -NHS. The differences were within 5%. In the pre-activation step, a wide range of molar excess of N 3 -NHS (four to fivefold excess) were mixed with 2 mg/mL of antibody or 1.2 mg/mL of apomyoglobin to achieve various I.R. After a two hours incubation at room temperature, the unreacted N 3 -NHS were removed by passing the samples twice through Zeba spin desalting columns. Attachment of an azido group to the protein does not affect the absorption spectrum of the protein, thus the concentration of the Ab-N 3 or apoMb-N 3 is determined with Ab's or apoMb's extinction coefficient (Ab: ε 280 = 217,500/M/cm, apoMb: ε 280 = 15,900/M/cm).

Figure 1.
Scheme for click labeling with targeted aDoL. The number in parentheses represents the molar ratio in the reaction. Using 15X molar excess of N 3 -NHS will usually yield ~ 5 N 3 -per protein provided the protein has sufficient lysine residues. The average degree of labeling (aDoL) can be controlled with the reagents' input molar ratio in the second reaction step. The tag can be DBCO, or other cyclooctynes complementary to N 3 -. Labeling protein with fluorophores-DBCO. All fluorophore-DBCO stocks were dissolved in DMSO to a final concentration of 10 mg/mL, then diluted in PBS buffer to ~ 100 μM. The 2 mg/mL Ab-N 3 (I.R. 7) was divided into small aliquots (50 μL), each reacted with 2× molar equivalent of AZ405-DBCO, AZ430-DBCO, AZ488-DBCO, AZ546-DBCO, AZ568-DBCO, AZ594-DBCO, or Cy5-DBCO. The products were analyzed on HPLC to determine reaction efficiencies.
Monitoring the click reaction by HPLC. The reaction kinetics of protein-N 3 and AZ488-DBCO was monitored by analytical chromatography using the Agilent Chemstation. 50 μL of the mixture was injected onto the analytical SRT-C SEC 300 (HPLC column, Sepax) at various time (5 min-24 h). Changes in the elution profile reflect the progress of the reaction. In the experiment, where 2 mg/mL of Ab-N 3 was reacted with nine different fluorophore-DBCOs, the reaction mixtures were injected onto the same column after a 24 ± 1.5-h incubation. There were 3 h of difference between the first and last sample injection. The detector was set at the absorption maximum of the corresponding fluorophore. The elution profiles were used to detect unreacted fluorophore-DBCO.
Fluorescence correlation spectroscopy (FCS). FCS experiments were performed using fluorescence correlation spectrometer (ALBA, ISS, Champaign, IL) integrated with an inverted Nikon Eclipse TE300 fluorescence microscope (Nikon InsTech Co., Ltd., Kanagawa, Japan) and a Spectra-Physics Mai Tai Ti-Sapphire laser 23 . The system is calibrated with analytically prepared 20 nM AlexaFluor 488 before each use. All samples were diluted to 50-100 nM in 10 mM HEPES buffer (pH 7.4, containing 0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant P20) and loaded in 384-well glass bottom plate for FCS measurement.

Impact of -N 3 labeling on antibody binding activities.
The antibodies used in the study are anti-NGAL antibody and anti-biotin antibody. Two independent protocols were used to compare the antibody's binding activity. In one approach, serial titrations were performed on anti-NGAL Ab 1 with three different IRs (I.R. 7, 15, and 24) and unlabeled Ab 1. The Abs were diluted to 100 pM in 10 mM HEPES buffer. Each Ab was titrated with a serial of 2× diluted NGAL-Cy5 solution. After overnight incubation, the antibody or antibody-NGAL-Cy5 complex was captured by secondary antibody (goat-anti-mouse IgG Ab) coated microparticles, the anti-NGAL Abs used in the study are mouse antibodies. Fluorescence signal from NGAL-Cy5 captured on the microparticles were used to compare the binding activities of three Ab-N 3 to unlabeled Ab. In the second approach, four different anti-NGAL antibodies and one anti-biotin antibody were labeled with N 3 -NHS with different I.R.s ranging from 2 to 6. All labeled antibodies and their corresponding intact antibodies were diluted to 100 pM in 10 mM HEPES buffer. 100 μL of each sample first reacted with 5 μL 0.1% solid NGAL coated microparticles for 30 min and then 15 min of 50 μL 30 nM Goat-anti-Mouse Antibody F(ab') 2 -Dylight649. The microparticles were washed after each binding reaction step, and then imaged on an Olympus epifluorescence microscope (instrument setup and image analysis were previous described 24,25 ). The biotin-NGAL coated microparticles were prepared by mixing 10 μL 15 μM biotin-NGAL, to 1 mL 0.1% solid streptavidin microparticles. Unbound biotin-NGAL were washed away from microparticle solution before each experiment using plate washer. The streptavidin coated microparticles were prepared by coupling 0.1 mg/mL streptavidin to 5μm paramagnetic carboxyl particles (Polymer Lab, Palo Alto, CA) kept at 1% (w/w) solids in the presence of 0.15 mg/mL EDAC (1-ethyl-3-(3-imethylaminpropyl) carbodiimide, hydrochloride. Streptavidin microparticles without the coating of biotin-NGAL was used as a negative control to ensure the measured signals were specific binding between the antibody and its corresponding antigen.

Results
Multiple labeling conditions and labeling products were described in this study, the following nomenclatures were used for clarity: protein-15×-N 3 -2×-AZ488, indicating the protein first reacts with 15× molar equivalent of N 3 -NHS, and then 2× molar equivalent of AZ488-DBCO. The number of N 3 -attached to the protein is referred to as the incorporation ratio (I.R.). The number of final fluorophores attached to the protein is referred as the average degree of labeling (aDoL). The intermediate product is referred as Protein-N 3 , the final product with fluorophores will be termed as the Conjugate.
Labeling antibody with AZ488-DBCO at an aDoL of 2. In the first example, the antibody was activated with 16× molar excess of N 3 -NHS, achieving an I.R. of 5.6. Then a 2:1 molar ratio of AZ488-DBCO and Ab-16×-N 3 were mixed to achieve an aDoL of 2. The reaction mixture (Ab-16×-N 3 , AZ488-DBCO) was injected onto the analytical HPLC column at various times to monitor the reaction progress and the product Ab-AZ488 was collected for further analysis. Figure 2a shows examples of chromatograms monitored at 493 nm over time.  Figure 2b shows the kinetic trace of the reaction extracted from the elution peak value at 8.8 min. The kinetic trace was then fit using Eq. (2) derived from the integrated rate equation for second order reaction (Eq. 1).
where [A] 0 and [B] 0 are the initial concentrations of N 3 -DBCO, and AZ488-DBCO, respectively; t is the reaction time in seconds, x is the concentration of AZ488-DBCO attached to the protein, and K is the reaction rate constant. The fitted K is 4.31 ± 0.13/M/s. We theoretically and experimentally confirmed that the click reaction was 100% complete after 24-h incubation provided the DBCO-tag and N 3 -tag were at minimal concentration of 10 μM and 25 μM, respectively. However, we did observe changes of the reaction rate constant or incomplete reaction on the second step depending www.nature.com/scientificreports/ on the size of the payload and targeted DoL, which might require longer incubation or target at a lower aDoL (see Supplementary section). Figure 2c shows the absorption spectra of the conjugates (from various incubation lengths) at the 8.8 min elution point. The increased 495 nm peak indicates more AZ488 were attaching to the antibody over time. The inset table lists the aDoL calculated from each absorption spectrum using the equation described below. After 24 h of incubation, the conjugate achieved an aDoL of 1.8, which is close to the target value of 2.
In parallel, FCS measurements were used to follow the progress of the reaction as well. The reaction mixtures were measured on the microscope without any purification step. Figure 2d shows the autocorrelation curves of the reaction mixture at various reaction time. The right shifted autocorrelation curves indicate more AZ488-DBCO are attached to the protein over time. At 24 h, the autocorrelation curve of the non-purified reaction mixture is superimposed with that of the HPLC purified conjugate, indicating all AZ488-DBCO had reacted with Ab-N 3 . Thus, we have used two independent methods to confirm the 100% completion of the labeling reaction.
Microscale and parallel labeling. 0.5 mg of Ab-17×-N 3 was divided into 9 aliquots and each reacted with 2× molar equivalents of AZ405-DBCO, AZ430-DBCO, AZ488-DBCO, AZ546-DBCO, AZ568-DBCO, AZ594-DBCO and Cy5-DBCO. All products were loaded onto the analytical HPLC column after ~ 24 h of incubation and were monitored at maximum absorption wavelength of each corresponding fluorophore. Unreacted residuals were found only in samples AZ532-DBCO and AZ647-DBCO. The rest all fully reacted with the protein (Fig. 3). We were unsure the cause of the incomplete reaction for AZ532-DBCO and AZ647-DBCO, but those two compounds should be avoided. The purpose of this experiment was to demonstrate that, after the activation step, the protein-N 3 can be conjugated with fluorophores, biotin or other small payloads with DBCO tag in parallel at microscales without additional need of purification, but one should confirm 100% reactivity of the DBCO-reagent with N 3 -tag during initial testing.
Comparison of the stability of attached fluorophore via click labeling or direct active ester-fluorophore labeling. Previously, when labeling proteins with AF488-SDP or AF488-NHS, free fluorophores were always detected in the conjugate solution after storing for only a few days. It has been suggested that the fluorophores were non-covalently attached to protein during the labeling reaction, and then would dissociate from the protein slowly over time, resulting in free fluorophore present in the purified protein conjugate. We feel N 3 -NHS should be less of a problem in that respect. The conjugated Ab-AF488 (direct attached through SDP ester functional group) and Ab-17×-N3-2×-AZ488 were prepared on the same day and measured on day 1 and day 100 using the FCS instrument. After 100 days at 2-8 °C, 15% of free AF488 was detected in the Ab-SDP-AF488 conjugate, while the conjugate labeled via SPAAC reaction had no free fluorophores detected (see S Fig. 5). This direct comparison experiment indicated that our click labeling approach can produce more stable conjugate, which can be explained by non-covalently bound fluorophore-DBCO (if any) can always react with protein-N 3 over time).
Labeling antibodies over a wide range of targeted aDoLs. The antibody was first reacted with 17×, 34× and 51× molar excess of N 3 -NHS, and achieved an I.R. of 7, 15, and 24 respectively. The protein-N 3 was then  www.nature.com/scientificreports/ reacted with AZ488-DBCO at molar ratios of 2, 4, 6, and 8. After 24-h incubation, the samples were diluted for fluorescence correlation spectroscopy (FCS) measurements without purification. As expected, AZ488-DBCO reacted 100% with Ab-17×-N 3 (I.R. 7) at aDoL target of 2 and 4, but trace amounts of AZ488-DBCO was detected for Ab-17×-N 3 (I.R. 7) at aDoL target of 6 and 8. On the other hand, Ab-34×-N 3 (I.R. 15) could accommodate up to 6 AZ488 per molecule, and Ab-51×-N 3 (I.R. 24) could accommodate up to 8 AZ488 per molecule (see S Fig. 4). A similar reaction protocol was performed on apoMb and achieved the targeted aDoL as expected (see S Fig. 3). This study suggests the I.R. of the protein-N 3 should be twice of the targeted aDoL to ensure the completion of the click reaction. However, it is known that the fluorescence brightness of labeled protein is not always linearly proportional to the number of fluorophores attached to it due to self-quenching, or sometimes referred to as concentration quenching 26,27 . We observed the expected self-quenching phenomena of the labeled protein with the FCS measurements. Figure 4 shows at aDoL of 2, the brightness of the conjugate is twice that of a single fluorophore AZ488-DBCO. There are incremental increases of brightness with higher aDoL, but it does not follow the linear trend. At aDoL of 8, the brightness of the conjugate is merely 3 times the brightness of fluorophore AZ488. Considering the potentially negative impact of extensive labeling to the protein's structure and function, and the minimal gain in brightness with higher aDoL 28 , it is important to emphasize that the aDoL should be kept low. Assuming the number of fluorophores per antibody follows a Poisson distribution 18,29 , even at aDoL of ~ 2, there will be ~ 18% of the protein with 3 labels, and ~ 15% of the protein with 4 and above labels.
Determination of I.R. and labeling efficiency for protein -N 3 . The functional tag, N 3 -does not absorb at 260 nm nor at 280 nm, thus attachment of N 3 -to the protein cannot be confirmed by absorption spectrum. Proteins with different I.R has very similar absorption spectra (see S Fig. 1). We have adopted two methods to determine the incorporation ratio of the N 3 -to protein. In one approach, the protein-N 3 reacts with excess molar of Cy5-DBCO, when every N 3 tag has a Cy5-DBCO attached, the aDoL of Cy5 to protein-N 3 should reflect the I.R. of Ab-N 3 and the aDoL can be determined by absorption spectra. This approach is more suitable for larger protein (e.g., Ab), which has a higher extinction coefficient and sufficient space to accommodate the fluorophores. Supplementary Table 1 listed the peak value and calculated aDoL of Ab-N 3 -DBCO-Cy5. An alternative approach is to use ESI-MS and directly resolve the number of N 3 -by the mass increase of the protein. This is more suited for smaller size protein without glycosylation (e.g., ApoMb). ESI-MS Spectra of apoMb-N 3 are included in Supplementary section (S Fig. 2). Table 1 lists the I.R. of Ab-N 3 and apoMb-N 3 , both methods show the reaction efficiency falls in the range of 25-47%. Excluding the over-labeled cases, the reaction efficiencies are at ~ 30% with the I.R. levels below 5.

Impact of N 3 labeling to antibody activity.
Two independent protocols were used to compare the antibody's binding activity upon -N 3 modification. In the first approach, binding titration curves were performed on anti-NGAL Ab1 at IRs of 0, 7, 15 and 24. Each binding curve has 10 data points. All binding curves are superimposable, indicating no impact of N 3 modification to the Antibody's function (Fig. 5a). In the second approach, we adopted a simpler method to evaluate more antibodies with different IRs (see "Materials and methods" section). All reagent (NGAL coated microparticles, goat-anti-mouse Ab-Dylight 649) concentrations and reaction conditions were kept same. Figure 5b shows the fluorescence signal of the original and N 3 labeled antibodies when binding to their corresponding antigen. The five antibodies have inherent different affinity toward its targeted antigen, which are reflected in the varied signal levels for each antibody. However, the signal variation from the same antibody labeled with different I.R. reflects the impact of labeling modification. Figure 5c shows normalized signal of labeled Abs to intact Ab. Anti-NGAL Ab1 and Ab4 showed the attachment of N 3 have no negative impact on the proteins' function, while anti-NGAL Ab2, Ab3 and anti-biotin Ab showed up to 30% binding activity loss at higher IRs. And there is an increase trend of functional loss with increased IR. The nega-  www.nature.com/scientificreports/ tive control experiment (Streptavidin coated microparticles) indicated all antibodies bind to NGAL or biotin specifically, the non-specific-binding signal at 100 pM Ab level is negligible.

Discussion
The labeling technique presented here is straightforward and precise. We are not aware of any existing labeling method which has the same features without performing chemo or site-selective labeling. As mentioned in the introduction, the major drawback of using active ester-fluorophore for protein conjugation is the inability to precisely control aDoL due to the instability of active ester-fluorophore and unpredictable reaction efficiency. Not all active ester tags are created equal. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDye® are particularly hygroscopic, further worsening the hydrolysis of active esters. In one incidence, we detected 37% of hydrolyzed Alexa Fluor® 488-NHS in a freshly opened vial by LC-MS (see S Fig. 5). The manufacture manual also acknowledged the % of active reagent are usually > 50%, but can be as low as 30-40%. With unknown level of hydrolysis, and the bulky size of the fluorophores, it is difficult to predict the reaction efficiency. Based on our experience, the labeling efficiency of AF488-NHS or AF488-SDP to protein can vary from 5 to 50%. Whereas N 3 -NHS is small, the intact N 3 -NHS and hydrolyzed N 3 -NHS have different absorption spectra, which could be used to check the integrity of the labeling reagent 22 . We tested various labeling ratios of N 3 -NHS to apoMb and antibody, the reaction efficiencies were at ~ 30% for both proteins with the I.R. below 5. We don't expect 30% reaction efficiency for every protein and every lot of N 3 -NHS, but it does show consistency and reproducibility. Furthermore, in a case of over-labeling, each additional N 3 -attachment is only 83 Dalton, it would have less impact to the protein's function comparing to a bulky fluorophore. The utility of this method was further demonstrated by the parallel labeling of 9 different fluorophores to the same protein-N 3 in a batch mode approach. After the activation step, the protein-N 3 can be conjugated with fluorophores, biotin or other small payloads with DBCO tag in parallel at microscales without additional need of purification. In a recently published paper 30 , the author tried to evaluate the effect of antibody biotinylation on an immunoassay using various linker lengths and various of aDoL on three antibodies. In total, 150 labeling Table 1. Labeling antibody and apoMb with N 3 -NHS at various ratios.